Adenosine released from underperfused or hypoxic cardiac myocytes is an important extracellular messenger. However, the specific mechanism responsible for increasing adenosine formation remains unclear. In prior studies we noticed an excellent correlation between adenosine release (Rado) and inorganic phosphate (Pi). This study tested the hypothesis that Pi increases Rado by inhibiting adenosine kinase (AK). AK was isolated from guinea pig hearts using a 5-AMP Sepharose affinity column. AK activity (V) was measured by conversion of labelled adenosine to AMP inhibitor iodotubercidin (20 mM). Pi concentrations seen during hypoxia or underperfusion substantially reduced the apparent Vmax. This demonstrates for the first time that Pi is an important regulator of cardiac adenosine formation via inhibition of adenosine kinase.